Fatty acid treatment for cardiac patients

ABSTRACT

A method of treating patients in need of treatment for a cardiac disorder has been found which comprises administering to the patient a seven carbon fatty acid compound or derivative thereof, wherein the compound or derivative thereof is able to readily enter the mitochondrion without special transport enzymes. A dietary formulation suitable for treatment of heart tissue in cardiac or surgical patients has been found which comprises a seven-carbon fatty acid chain, wherein the seven-carbon fatty acid chain is characterized by the ability to transverse the inner mitochondrial membrane by a transport mechanism which does not require carnitine palmitoyltransferase I, carnitine palmitoyltransferase II, or carnitine/acylcarnitine translocase and the ability to undergo mitochondrial β-oxidation, and wherein the compound is selected from the group consisting of n-heptanoic acid or a derivative thereof, a triglyceride comprising n-heptanoic acid or a derivative thereof, and triheptanoin or a derivative thereof.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of copending U.S. application Ser. No.10/371,685 filed on 21 Feb. 2003 which is a divisional of copending U.S.application Ser. No. 09/890,559 filed on 1 Aug. 2001 which claims thebenefit of U.S. Provisional Application No. 60/119,038 which was filedon 5 Feb. 1999.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a nutritional or dietetic composition orsupplement.

BACKGROUND OF THE INVENTION

Fatty acid oxidation plays a major role in the production of energy, andis essential during periods of fasting. Serious disorders in fatty acidmetabolism can arise which range from skeletal and/or cardiac muscleweakness to episodes of metabolic apnea to death resembling suddeninfant death syndrome. These disorders manifest with severecardiomyopathy, hypoglycemia, myopathy, microvesicular fat deposition inaffected organs, and/or fulminant hepatic failure. Patients sufferingfrom inborn genetic errors in fatty acid metabolism often experiencefatal or repeated severely debilitating episodes upon failure togenerate energy via fatty acid metabolism. Premature infants require amaintenance of a high blood sugar level. Often, their routine diet doesnot provide sufficient amounts of carbohydrate energy sources and theirfat metabolism enzymes are not efficient at birth. Elderly patients alsoexperience difficulty in the regulation of blood sugar levels due todecreased appetite and inefficient metabolism.

Saturated fatty acids are represented by the following structure

where R represents an alkyl group. Naturally occurring fatty acidsderived from higher plant and animal lipids include both saturated andunsaturated even-numbered carbon chains. The most abundant naturallyoccurring saturated fatty acids are palmitic acid (16 carbons; C₁₆) andstearic acid (18 carbons; C₁₈). Shorter-chain fatty acids (12-14carbons; C₁₂ to C₁₄) and longer-chain fatty acids (up to 28 carbons;C₂₈) naturally occur in small quantities. Fatty acids of less than 10carbons are rarely present in animal lipids, with the exception of milkfat comprising about 32% oleic acid (unsaturated C₁₈), about 15%palmitic acid (C₁₆), about 20% myristic acid (C₁₄), about 15% stearicacid (C₁₈), about 6% lauric acid (C₁₂), and about 10% fatty acids of4-10 carbons (C₄-C₁₀).

Fatty acids are generally categorized by the length of the carbon chainattached to the carboxyl group: short-chain for 4 to 6 carbons (C₄-C₆),medium-chain for 8 to 14 carbons (C₈-C₁₄), long-chain for 16 to 18carbons (C₁₆-C₁₈), and very long-chains for 20 to 28 carbons (C₂₀-C₂₈).

The process by which fatty acids are metabolized involves mitochondrialβ-oxidation in the mitochondria of the cell. As illustrated in FIG. 1,fatty acid oxidation of a long-chain fatty acid such as palmitic acidbegins transport of the fatty acid through the plasma membrane via aplasma membrane carnitine transporter. As the fatty acid passes throughthe outer mitochondrial membrane, the fatty acid is converted in thepresence of Coenzyme A (CoASH) and acyl-CoA synthetase into a fatty acidester of Coenzyme A (fatty acyl-CoA) at the expense of ATP. The fattyacyl-CoA is converted into fatty acylcarnitine in the presence ofcarnitine and carnitine palmitoyltransferase I (CPT I). The fattyacylcarnitine then passes the inner membrane of the mitochondria, a stepwhich is catalyzed by the carnitine/acylcarnitine translocase enzyme.Once inside the mitochondria, the fatty acylcarnitine is then convertedback into fatty acyl-CoA in the presence of carnitinepalmitoyltransferase II (CPT II). In the oxidation cycle within themitochondria, the fatty acyl-CoA is dehydrogenated by removal of a pairof hydrogen atoms from the α and β carbon atoms via a chain-specificacyl-CoA dehydrogenase to yield the α,β-unsaturated acyl-CoA, or2-trans-enoyl-CoA. The appropriate acyl-CoA dehydrogenase is determinedby the carbon chain length of the fatty acyl-CoA, i.e., long-chainacyl-CoA dehydrogenase (LCAD; C₁₂ to C₁₈), medium-chain acyl-CoAdehydrogenase (MCAD; C₄ to C₁₂), short-chain acyl-CoA dehydrogenase(SCAD; C₄ to C₆), or very long-chain acyl-CoA dehydrogenase (VLCAD; C₁₄to C₂₀). The α,β-unsaturated acyl-CoA is then enzymatically hydrated via2-enoyl-CoA hydratase to form L-3-hydroxyacyl-CoA, which in turn isdehydrogenated in an NAD-linked reaction catalyzed by a chain-specificL-3-hydroxyacyl-CoA dehydrogenase to form β-ketoacyl-CoA. Theappropriate L-3-hydroxyacyl-CoA dehydrogenase is determined by thecarbon chain length of the L-3-hydroxyacyl-CoA, i.e., long-chainL-3-hydroxyacyl-CoA dehydrogenase (LCHAD; C₁₂ to C₁₈) or short-chainL-3-hydroxyacyl-CoA dehydrogenase (SCHAD; C₄ to C₁₆ with decreasingactivity with increasing chain length). The β-ketoacyl CoA esterundergoes enzymatic cleavage by attack of the thiol group of a secondmolecule of CoA in the presence of 3-ketoacyl-CoA thiolase, to formfatty acyl-CoA and acetyl-CoA derived from the α carboxyl and the βcarbon atoms of the original fatty acid chain. The other product, along-chain saturated fatty acyl-CoA having two fewer carbon atoms thanthe starting fatty acid, now becomes the substrate for another round ofreactions, beginning with the first dehydrogenation step, until a secondtwo-carbon fragment is removed as acetyl-CoA. At each passage throughthis spiral process, the fatty acid chain loses a two-carbon fragment asacetyl-CoA and two pairs of hydrogen atoms to specific acceptors.

Each step of the fatty acid oxidation process is catalyzed by enzymeswith overlapping carbon chain-length specificities Inherited disordersof fatty acid oxidation have been identified in association with theloss of catalytic action by these enzymes. These include defects ofplasma membrane carnitine transport; CPT I and II;carnitine/acylcarnitine translocase; very-long-chain, medium-chain, andshort-chain acyl-CoA dehydrogenases (i.e., VLCAD, MCAD, and SCAD,respectively); 2,4-dienoyl-CoA reductase; long-chain 3-hydroxyacyl-CoAdehydrogenase acyl-CoA (LCHAD), and mitochondrial trifunctional protein(MTP) deficiency. To date, treatment for medium chain dehydrogenase(MCAD) deficiency has been found. However, the remaining defects oftenare fatal to patients within the first year of life, and no knowneffective treatment has been made available. In particular, patientssuffering from severe carnitine/acylcarnitine translocase deficiencyroutinely die, there are no known survivors, and no known treatment hasbeen found.

Attempts to treat these disorders have centered around providing foodsources which circumvent the loss of catalytic action by the defectiveenzyme. For example, the long-chain fatty acid metabolic deficiencycaused by a defective carnitine/acylcarnitine translocase enzyme(referred hereinafter as “translocase deficiency”) often leads to deathin the neonatal period. Providing carnitine, a high carbohydrate diet,and medium-chain triglycerides to one translocase-deficient patientfailed to overcome the fatty acid metabolic deficiency. It was believedthat the metabolism of medium-chain fatty acids would not require thecarnitine/acylcarnitine translocase enzyme, since medium-chain fattyacids are expected to freely enter the mitochondria. Thus, infantformulas were developed comprising even-carbon number medium-chaintriglycerides (MCT) (e.g., 84% C₈, 8% C₆ and 8% C₁₀) which were expectedto by-pass the translocase defect. Fatalities continue to occur despitetreatment attempts with these formulas.

With the exception of pelargonic acid (saturated fatty acid with 9carbons; C₉), odd-carbon number fatty acids are rare in higher plant andanimal lipids. Certain synthetic odd-carbon number triglycerides havebeen tested for use in food products as potential fatty acid sources andin the manufacture of food products. The oxidation rates of odd-chainfatty acids from C₇ and C₉ triglycerides have been examined in vitro inisolated piglet hepatocytes. (Odle, et al. 1991. “Utilization ofmedium-chain triglycerides by neonatal piglets: chain length of even-and odd-carbon fatty acids and apparent digestion/absorption and hepaticmetabolism,” J Nutr 121:605-614; Lin, X, et al. 1996. “Acetaterepresents a major product of heptanoate and octanoate beta-oxidation inhepatocytes isolated from neonatal piglets,” Biochem J 318:235-240; andOdle, J. 1997. “New insights into the utilization of medium-chaintriglycerides by the neonate: observations from a piglet model,” J Nutr127:1061-1067). The importance of odd-chain fatty acids propionate (C₃),valerate (C₅), and nonanoate (C₉) as gluconeogenic precursors wasevaluated in hepatocytes from starved rats. (Sugden, et al. 1984.“Odd-carbon fatty acid metabolism in hepatocytes from starved rats,”Biochem Int'l 8:61-67). The oxidation of radiolabeled margarate (C₁₇)was examined in rat liver slices. (Boyer, et al. 1970. “Hepaticmetabolism of 1-¹⁴C octanoic and 1-¹⁴C margaric acids,” Lipids4:615-617).

In vivo studies utilizing C₃, C₅, C₇, C₉, C₁₁, and C₁₇ have also beencarried out in vivo in guinea pigs, rabbits, and rats. In vivo oxidationrates of systematically infused medium-chain fatty acids from C₇ and C₉triglycerides, and a C₇/C₉ triglyceride mixture have been examined inneonatal pigs. (Odle, et al. 1992. “Evaluation of [1-14C]-medium-chainfatty acid oxidation by neonatal piglets using continuous-infusionradiotracer kinetic methodology,” J Nutr 122:2183-2189; and Odle, et al.1989. “Utilization of medium-chain triglycerides by neonatal piglets:II. Effects of even- and odd-chain triglyceride consumption over thefirst 2 days of life on blood metabolites and urinary nitrogenexcretion,” J Anima Sci 67:3340-3351). Rats fed triundecanoin (saturatedC₁₁) were observed to maintain nonfasting blood glucose levels duringprolonged fasting. (Anderson, et al. 1975. “Glucogenic and ketogeniccapacities of lard, safflower oil, and triundecanoin in fasting rats,” JNutr 105:185-189.) An emulsion of trinonanoin (C₉) and long-chaintriglycerides was infused into rabbits for evaluation as long-term totalparenteral nutrition. (Linseisen, et al. 1993. “Odd-numberedmedium-chain triglycerides (trinonanoin) in total parenteral nutrition:effects on parameters of fat metabolism in rabbits,” J Parenteral andEnteral Nutr 17:522-528). The triglyceride triheptanoin containing thesaturated 7-carbon fatty n-heptanoic acid (C₇) has also been reportedlyused in Europe in agricultural feed, as a tracer molecule in themanufacture of butter, and as a releasing agent in the manufacture ofchocolates and other confectionaries. However, there has been noindication heretofore that a seven-carbon fatty acid is safe forconsumption by humans or has any particular nutritional benefit tohumans.

It has now been found that acquired metabolic derangements and inheritedmetabolic disorders, especially fatty acid metabolic defects, can beovercome using a nutritional composition comprising a seven-carbon fattyacid (C₇) such as n-heptanoic acid. Patients experiencing defective orreduced fatty acid metabolism can be treated with a nutritionalcomposition comprising a seven-carbon fatty acid such as n-heptanoicacid and/or its triglyceride triheptanoin as a very efficient energysource. Patients needing rapid energy may also benefit from consumptionof the seven-carbon fatty acid or its triglyceride.

SUMMARY OF THE INVENTION

In one aspect, the present invention is a method of treating patients inneed of treatment for a cardiac disorder, comprising administering tothe patient a seven carbon fatty acid compound or derivative thereof,wherein the compound or derivative thereof is able to readily enter themitochondrion without special transport enzymes. In a preferred method,the seven carbon fatty acid compound comprises n-heptanoic acid. Inanother preferred method, the seven carbon fatty acid compound comprisesa triglyceride comprising n-heptanoic acid, for example, triheptanoin.In a preferred method, the derivative is a five carbon fatty acid chain.In another preferred method, the derivative is selected from the groupconsisting of 4-methylhexanoate, 4-methylhexenoate,3-hydroxy-4-methylhexanoate, 5-methylhexanoate, 5-methylhexenoate and3-hydroxy-5-methylhexanoate. In a preferred method, the compound orderivative thereof is capable of being broken down by normal β-oxidationin humans to methylbutyric acid. In another preferred method, thecompound or derivative thereof is capable of being broken down by normalβ-oxidation in humans to isovaleric acid. In another preferred method,the compound or derivative is capable of being broken down by normalβ-oxidation in humans to n-valeryl-CoA. In yet another preferred method,the compound or derivative is capable of being broken down by normalβ-oxidation in humans to propionyl-CoA in one or more oxidativeprocedures. Preferably, the compound or derivative thereof is providedto the patient in an amount comprising at least about 25% of the dietarycaloric requirement for the patient. Preferably, the compound orderivative is provided orally, parenterally, or intraperitoneally. Thismethod is suitable for treating cardiac disorders such as cardiacmyopathy. It is also suitable for treatment of the aftermath of heartsurgery, wherein the compound or derivative is utilized for directfueling of heart muscle.

In another aspect, the present invention is a dietary formulationsuitable for treatment of heart tissue in cardiac or surgical patientscomprising a seven-carbon fatty acid chain, wherein the seven-carbonfatty acid chain is characterized by the ability to transverse the innermitochondrial membrane by a transport mechanism which does not requirecarnitine palmitoyltransferase I, carnitine palmitoyltransferase II, orcarnitine/acylcarnitine translocase and the ability to undergomitochondrial β-oxidation, and wherein the compound is selected from thegroup consisting of n-heptanoic acid or a derivative thereof, atriglyceride comprising n-heptanoic acid or a derivative thereof, andtriheptanoin or a derivative thereof.

In another aspect, the present invention is a dietary formulationsuitable for treatment of heart tissue in cardiac or surgical patientscomprising a seven carbon fatty acid compound or derivative thereof,wherein the compound or derivative thereof is able to readily enter themitochondrion without special transport enzymes. In a preferred dietaryformulation, the seven carbon fatty acid compound comprises n-heptanoicacid. In another preferred dietary formulation, the seven carbon fattyacid compound comprises a triglyceride comprising n-heptanoic acid, forexample, triheptanoin. In a preferred dietary formulation, thederivative is a five carbon fatty acid chain. In another preferreddietary formulation, the derivative is selected from the groupconsisting of 4-methylhexanoate, 4-methylhexenoate,3-hydroxy-4-methylhexanoate, 5-methylhexanoate, 5-methylhexenoate and3-hydroxy-5-methylhexanoate. In a preferred dietary formulation, thecompound or derivative thereof is capable of being broken down by normalβ-oxidation in humans to methylbutyric acid. In another preferreddietary formulation, the compound or derivative thereof is capable ofbeing broken down by normal β-oxidation in humans to isovaleric acid. Inanother preferred dietary formulation, the compound or derivative iscapable of being broken down by normal β-oxidation in humans ton-valeryl-CoA. In yet another preferred dietary formulation, thecompound or derivative is capable of being broken down by normalβ-oxidation in humans to propionyl-CoA in one or more oxidativeprocedures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting the pathway of mitochondrial β-oxidationfor long-chain fatty acids, with the required transporters and enzymesitalicized and the three designated membranes represented by doublelines.

FIG. 2 is a diagram depicting the pathway of mitochondrial β-oxidationfor n-heptanoic acid, with the required transporters and enzymesitalicized and the designated inner mitochondrial membrane representedby a double line.

FIG. 3A is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a deceased child who suffered from severe translocasedeficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition.

FIG. 3B is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from the deceased child reported in FIG. 3A who suffered fromsevere translocase deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition.

FIG. 4A is a graph depicting a tandem mass spectrometry profile foramniocytes treated with D3-C16 (²H₃-palmitate-C16). The amniocytes wereobtained from a fetus diagnosed with severe translocase deficiency,whose sibling was the deceased child reported in FIGS. 3A and 3B. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition.

FIG. 4B is a graph depicting a tandem mass spectrometry profile foramniocytes treated with D3-C7 (7-²H₃-heptanoate). The amniocytes wereobtained from the fetus reported in FIG. 4A who was diagnosed withsevere translocase deficiency and whose sibling was the deceased childreported in FIGS. 3A and 3B. Test parameters were: parents of 99FB (fastatom bombardment) and MCA acquisition.

FIG. 5A is a graph depicting a tandem mass spectrometry profile fornormal fibroblasts treated with D3-C7 (7-²H₃-heptanoate). Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. Internal standards for the profiles in FIG. 5A-5C arelocated at m/z 420.3 (²H₆-palmitate-C16), m/z 308.2 (²H₆-octanoate-C8),m/z 269.1 (²H₉-isovaleric-C5), and m/z 237.0 (²H₅-propionate-C3),wherein m/z is the mass:charge ratio. The peak at m/z 291 representsD3-C7 (7-²H₃-heptanoate). The peak at m/z 235 represents D3-C3(3-²H₃-propionate), the end point of odd-carbon degradation.

FIG. 5B is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from carnitine palmitoyltransferase I(CPT I) deficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition. The peak at m/z 291 represents D3-C7(7-²H₃-heptanoate). The peak at m/z 235.0 represents D3-C3(3-²H₃-propionate), the end point of odd-carbon degradation.

FIG. 5C is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from translocase deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 291.3 represents D3-C7 (7-²H₃-heptanoate).The peak at m/z 235 represents D3-C3 (3-²H₃-propionate), the end pointof odd-carbon degradation.

FIG. 5D is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from carnitine palmitoyltransferaseII (CPT II) deficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition. Internal standards for the profiles inFIG. 5D-5F are located at m/z 420.4 (²H₆-palmitate-C16), m/z 308.3(²H₆-octanoate-C8), m/z 269.2 (²H₉-isovaleric-C5), and m/z 237.1(²H₅-propionate-C3). The peak at m/z 291.1 represents D3-C7(7-²H₃-heptanoate). The peak at m/z 235 represents D3-C3(3-²H₃-propionate), the end point of odd-carbon degradation.

FIG. 5E is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from the “cardiac” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-C) deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 291 represents D3-C7 (7-²H₃-heptanoate).The peak at m/z 235.1 represents D3-C3 (3-²H₃-propionate), the end pointof odd-carbon degradation.

FIG. 5F is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from the “hypoglycemic” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-H) deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 291.4 represents D3-C7 (7-²H₃-heptanoate).The peak at m/z 235.1 represents D3-C3 (3-²H₃-propionate), the end pointof odd-carbon degradation.

FIG. 5G is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from mitochondrial trifunctionalprotein (TRIFUNCTIONAL) deficiency. Test parameters were: parents of99FB (fast atom bombardment) and MCA acquisition. Internal standards forthe profiles in FIG. 5G-5I are located at m/z 420.3 (²H₆-palmitate-C16),m/z 308.1 (²H₆-octanoate-C8), m/z 269.0 (²H₉-isovaleric-C5), and m/z237.2 (²H₅-propionate-C3). The peak at m/z 291 represents D3-C7(7-²H₃-heptanoate). The peak at m/z 235.1 represents D3-C3(3-²H₃-propionate), the end point of odd-carbon degradation.

FIG. 5H is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from long-chain L-3-hydroxy-acyl-CoAdehydrogenase (LCHAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. The peak at m/z 291.1represents D3-C7 (7-²H₃-heptanoate). The peak at m/z 235.1 representsD3-C3 (3-²H₃-propionate), the end point of odd-carbon degradation.

FIG. 5I is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from medium-chain acyl-CoAdehydrogenase (MCAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. The peak at m/z 291.2represents D3-C7 (7-²H₃-heptanoate). The peak at m/z 235 representsD3-C3 (3-²H₃-propionate), the end point of odd-carbon degradation.

FIG. 5J is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from short-chain acyl-CoAdehydrogenase (SCAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. Internal standards for theprofiles in FIG. 5J-5L are located at m/z 420.4 (²H₆-palmitate-C16), m/z308.0 (²H₆-octanoate-C8), m/z 269.2 (²H₉-isovaleric-C5), and m/z 237(²H₅-propionate-C3). The peak at m/z 291.1 represents D3-C7(7-²H₃-heptanoate). The peak at m/z 235.1 represents D3-C3(3-²H₃-propionate), the end point of odd-carbon degradation.

FIG. 5K is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from electron transfer flavoproteinQO dehydrogenase-mild (ETF-DH mild) deficiency. Test parameters were:parents of 99FB (fast atom bombardment) and MCA acquisition. The peak atm/z 291.3 represents D3-C7 (7-²H₃-heptanoate).

FIG. 5L is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C7 (7-²H₃-heptanoate). The fibroblasts wereobtained from a child who suffered from electron transfer flavoproteinQO dehydrogenase-severe (ETF-DH severe) deficiency. Test parameterswere: parents of 99FB (fast atom bombardment) and MCA acquisition. Thepeak at m/z 291 represents D3-C7 (7-²H₃-heptanoate).

FIG. 6A is a graph depicting a tandem mass spectrometry profile fornormal fibroblasts treated with D3-C8 (8-²H₃-octanoate). Test parameterswere: parents of 99FB (fast atom bombardment) and MCA acquisition.Internal standards for the profiles in FIG. 6A-6C are located at m/z420.4 (²H₆-palmitate-C16), m/z 308.3 (²H₆-octanoate-C8), m/z 269.2(²H₉-isovaleric-C5), and m/z 237.1 (²H₅-propionate-C3). The peak at m/z305.3 represents D3-C8 (8-²H₃-octanoate).

FIG. 6B is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from carnitine palmitoyltransferase I(CPT I) deficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition. The peak at m/z 305.0 represents D3-C8(8-²H₃-octanoate).

FIG. 6C is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from translocase deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 305.3 represents D3-C8 (8-²H₃-octanoate).

FIG. 6D is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from carnitine palmitoyltransferaseII (CPT II) deficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition. Internal standards for the profiles inFIG. 6D-6F are located at m/z 420.3 (²H₆-palmitate-C16), m/z 308.3(²H₆-octanoate-C8), m/z 269.2 (²H₉-isovaleric-C5), and m/z 237.2(²H₅-propionate-C3). The peak at m/z 305.3 represents D3-C8(8-²H₃-octanoate).

FIG. 6E is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from the “cardiac” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-C) deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 305.3 represents D3-C8 (8-²H₃-octanoate).

FIG. 6F is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from the “hypoglycemic” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-H) deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 305.2 represents D3-C8 (8-²H₃-octanoate).

FIG. 6G is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from mitchodrial trifunctionalprotein (TRIFUNCTIONAL) deficiency. Test parameters were: parents of99FB (fast atom bombardment) and MCA acquisition. Internal standards forthe profiles in FIG. 6G-6I are located at m/z 420.5 (²H₆-palmitate-C16),m/z 308.3 (²H₆-octanoate-C8), m/z 269.2 (²H₉-isovaleric-C5), and m/z237.2 (²H₅-propionate-C3). The peak at m/z 305.3 represents D3-C8(8-²H₃-octanoate).

FIG. 6H is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from long-chain L-3-hydroxy-acyl-CoAdehydrogenase (LCHAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. The peak at m/z 305represents D3-C8 (8-²H₃-octanoate).

FIG. 6I is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from medium-chain acyl-CoAdehydrogenase (MCAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. The peak at m/z 305.2represents D3-C8 (8-²H₃-octanoate).

FIG. 6J is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from short-chain acyl-CoAdehydrogenase (SCAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. Internal standards for theprofiles in FIG. 6J-6L are located at m/z 420.4 (²H₆-palmitate-C16), m/z308.1 (²H₆-octanoate-C8), m/z 269.2 (²H₉-isovaleric-C5), and m/z 237(²H₅-propionate-C3). The peak at m/z 305.0 represents D3-C8(8-²H₃-octanoate).

FIG. 6K is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from electron transfer flavoproteinQO dehydrogenase-mild (ETF-DH mild) deficiency. Test parameters were:parents of 99FB (fast atom bombardment) and MCA acquisition. The peak atm/z 305.2 represents D3-C8 (8-²H₃-octanoate).

FIG. 6L is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C8 (8-²H₃-octanoate). The fibroblasts wereobtained from a child who suffered from electron transfer flavoproteinQO dehydrogenase-severe (ETF-DH severe) deficiency. Test parameterswere: parents of 99FB (fast atom bombardment) and MCA acquisition. Thepeak at m/z 305.3 represents D3-C8 (8-²H₃-octanoate).

FIG. 7A is a graph depicting a tandem mass spectrometry profile fornormal fibroblasts treated with D3-C9 (9-²H₃-nonanoate). Test parameterswere: parents of 99FB (fast atom bombardment) and MCA acquisition.Internal standards for the profiles in FIG. 7A-7C are located at m/z420.4 (²H₆-palmitate-C16), m/z 308.2 (²H₆-octanoate-C8), m/z 269.2(²H₉-isovaleric-C5), and m/z 237.3 (²H₅-propionate-C3). The peak at m/z319.3 represents D3-C9 (9-²H₃-nonanoate).

FIG. 7B is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from carnitine palmitoyltransferase I(CPT I) deficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition. The peak at m/z 319.3 represents D3-C9(9-²H₃-nonanoate).

FIG. 7C is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from translocase deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 319.3 represents D3-C9 (9-²H₃-nonanoate).

FIG. 7D is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from carnitine palmitoyltransferaseII (CPT II) deficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition. Internal standards for the profiles inFIG. 7D-7F are located at m/z 420.5 (²H₆-palmitate-C16), m/z 308.3(²H₆-octanoate-C8), m/z 269.3 (²H₉-isovaleric-C5), and m/z 237.1(²H₅-propionate-C3). The peak at m/z 319.3 represents D3-C9(9-²H₃-nonanoate).

FIG. 7E is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from the “cardiac” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-C) deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 319.3 represents D3-C9 (9-²H₃-nonanoate).

FIG. 7F is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from the “hypoglycemic” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-H) deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 319.3 represents D3-C9 (9-²H₃-nonanoate).

FIG. 7G is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from mitchondrial trifunctionalprotein (TRIFUNCTIONAL) deficiency. Test parameters were: parents of99FB (fast atom bombardment) and MCA acquisition. Internal standards forthe profiles in FIG. 7G-7I are located at m/z 420.5 (²H₆-palmitate-C16),m/z 308.2 (²H₆-octanoate-C8), m/z 269.3 (²H₉-isovaleric-C5), and m/z237.2 (²H₅-propionate-C3). The peak at m/z 319.3 represents D3-C9(9-²H₃-nonanoate).

FIG. 7H is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from long-chain L-3-hydroxy-acyl-CoAdehydrogenase (LCHAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. The peak at m/z 319.2represents D3-C9 (9-²H₃-nonanoate).

FIG. 7I is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from medium-chain acyl-CoAdehydrogenase (MCAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. The peak at m/z 319.0represents D3-C9 (9-²H₃-nonanoate).

FIG. 7J is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from short-chain acyl-CoAdehydrogenase (SCAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. Internal standards for theprofiles in FIG. 7J-7L are located at m/z 420.4 (²H₆-palmitate-C16), m/z308.2 (²H₆-octanoate-C8), m/z 269.3 (²H₉-isovaleric-C5), and m/z 237.0(²H₅-propionate-C3). The peak at m/z 319.3 represents D3-C9(9-²H₃-nonanoate).

FIG. 7K is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from electron transfer flavoproteinQO dehydrogenase-mild (ETF-DH mild) deficiency. Test parameters were:parents of 99FB (fast atom bombardment) and MCA acquisition. The peak atm/z 319.3 represents D3-C9 (9-²H₃-nonanoate).

FIG. 7L is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C9 (9-²H₃-nonanoate). The fibroblasts wereobtained from a child who suffered from electron transfer flavoproteinQO dehydrogenase-severe (ETF-DH severe) deficiency. Test parameterswere: parents of 99FB (fast atom bombardment) and MCA acquisition. Thepeak at m/z 319.3 represents D3-C9 (9-²H₃-nonanoate).

FIG. 8A is a graph depicting a tandem mass spectrometry profile fornormal fibroblasts treated with D3-C16 (16-²H₃-palmitate). Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. Internal standards for the profiles in FIG. 8A-8C arelocated at m/z 420.4 (²H₆-palmitate-C16), m/z 308.2 (²H₆-octanoate-C8),m/z 269.2 (²H₉-isovaleric-C5), and m/z 237.1 (²H₅-propionate-C3). Thepeak at m/z 417.0 represents D3-C16 (16-²H₃-palmitate).

FIG. 8B is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from carnitine palmitoyltransferase I(CPT I) deficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition. The peak at m/z 417.6 representsD3-C16 (16-²H₃-palmitate).

FIG. 8C is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from translocase deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 417.4 represents D3-C16 (16-²H₃-palmitate).

FIG. 8D is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from carnitine palmitoyltransferaseII (CPT II) deficiency. Test parameters were: parents of 99FB (fast atombombardment) and MCA acquisition. Internal standards for the profiles inFIG. 8D-8F are located at m/z 420.4 (²H₆-palmitate-C16), m/z 308.2(²H₆-octanoate-C8), m/z 269.2 (²H₉-isovaleric-C5), and m/z 237.2(²H₅-propionate-C3). The peak at m/z 417.4 represents D3-C16(16-²H₃-palmitate).

FIG. 8E is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from the “cardiac” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-C) deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 417.5 represents D3-C16 (16-²H₃-palmitate).

FIG. 8F is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from the “hypoglycemic” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-H) deficiency. Testparameters were: parents of 99FB (fast atom bombardment) and MCAacquisition. The peak at m/z 417.5 represents D3-C16 (16-²H₃-palmitate).

FIG. 8G is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from mitochondrial trifunctionalprotein (TRIFUNCTIONAL) deficiency. Test parameters were: parents of99FB (fast atom bombardment) and MCA acquisition. Internal standards forthe profiles in FIG. 8G-8I are located at m/z 420.5 (²H₆-palmitate-C16),m/z 308.3 (²H₆-octanoate-C8), m/z 269.2 (²H₉-isovaleric-C5), and m/z237.0 (²H₅-propionate-C3). The peak at m/z 417.4 represents D3-C16(16-²H₃-palmitate).

FIG. 8H is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from long-chain L-3-hydroxy-acyl-CoAdehydrogenase (LCHAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. The peak at m/z 417.4represents D3-C16 (16-²H₃-palmitate).

FIG. 8I is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from medium-chain acyl-CoAdehydrogenase (MCAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. The peak at m/z 417represents D3-C16 (16-²H₃-palmitate).

FIG. 8J is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from short-chain acyl-CoAdehydrogenase (SCAD) deficiency. Test parameters were: parents of 99FB(fast atom bombardment) and MCA acquisition. Internal standards for theprofiles in FIG. 8J-8L are located at m/z 420.4 (²H₆-palmitate-C16), m/z308 (²H₆-octanoate-C8), m/z 269.1 (²H₉-isovaleric-C5), and m/z 237(²H₅-propionate-C3). The peak at m/z 417 represents D3-C16(16-²H₃-palmitate).

FIG. 8K is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from electron transfer flavoproteinQO dehydrogenase-mild (ETF-DH mild) deficiency. Test parameters were:parents of 99FB (fast atom bombardment) and MCA acquisition. The peak atm/z 417.3 represents D3-C16 (16-²H₃-palmitate).

FIG. 8L is a graph depicting a tandem mass spectrometry profile forfibroblasts treated with D3-C16 (16-²H₃-palmitate). The fibroblasts wereobtained from a child who suffered from electron transfer flavoproteinQO dehydrogenase-severe (ETF-DH severe) deficiency. Test parameterswere: parents of 99FB (fast atom bombardment) and MCA acquisition. Thepeak at m/z 417.3 represents D3-C16 (16-²H₃-palmitate).

DETAILED DESCRIPTION

It has now been determined that fatty acids having seven carbons (C₇) ortheir triglycerides do not require the usual enzymes needed fortransporting long-chain fatty acids into the mitochondrion for energyproduction, i.e., carnitine/acylcarnitine translocase, carnitinepalmitoyltransferase (“CPT”) I and CPT II. Thus, triglycerides composedof seven-carbon fatty acids are useful as a nutritional supplement toovercome fatty acid metabolism deficiencies that require such enzymes.Nutritional supplements or pharmaceutical preparations comprisingseven-carbon fatty acids are useful in treatment of inherited metabolicdisorders as well as acquired metabolic derangements.

A preferred seven-carbon fatty acid is n-heptanoic acid. n-Heptanoicacid is a saturated straight chain seven-carbon fatty acid with thefollowing structure:

Triheptanoin is a triglyceride made by the esterification of threen-heptanoic acid molecules and glycerol. In regard to therapy, the termsheptanoic acid, heptanoate, and triheptanoin may be used interchangeablyin the following description. Also, it will be understood by thoseskilled in the art that heptanoic acid, heptanoate, and triheptanoin areused throughout the following description as an exemplary seven-carbonfatty acid source of the invention and is intended to be illustrative ofthe invention, but is not to be construed to limit the scope of theinvention in any way. Substituted, unsaturated, or branched heptanoate,as well as other modified seven-carbon fatty acids can be used withoutdeparting from the scope of the invention.

Triheptanoin is first broken down into three molecules of heptanoic acidand glycerol. As illustrated in FIG. 2, the heptanoic acid is thenbroken down through normal β-oxidative procedures to n-valeryl-CoA (C₅)and acetyl-CoA (C₂) in the first cycle. In the second cycle, then-valeryl-CoA is then broken down to propionyl-CoA (C₃) and acetyl-CoA(C₂), both of which are important precursors as fuel for the Kreb'scycle and energy production. Triheptanoin, therefore, is useful as anefficient source of fuel for energy production. Additionally,propionyl-CoA is a direct precursor for glucose production.Consequently, triheptanoin is useful as a dietary supplement forpatients susceptible to hypoglycemia, especially for premature infantsand the elderly. Triheptanoin can also be utilized as a growth ratestimulant for premature infants, allowing for shorter hospitalizationsand thereby reducing medical costs for these infants. Further, sincefatty acids are the major fuel for heart tissue and because it has theproperty of being gluconeogenic, triheptanoin can be used in directfueling of heart tissue in adults recuperating from cardiac or otherhigh-risk surgery.

Heptanoic acid is found in various fusel oils in appreciable amounts andcan be extracted by any means known in the art. It can also besynthesized by oxidation of heptaldehyde with potassium permanganate indilute sulfuric acid. (Ruhoff, Org Syn coll. vol II, 315 (1943)).Heptanoic acid is also commercially available through Sigma Chemical Co.(St. Louis, Mo.).

Triheptanoin can be obtained by the esterification of heptanoic acid andglycerol by any means known in the art. Triheptanoin is alsocommercially available through Condea Chemie GmbH (Witten, Germany) asSpecial Oil 107.

Unsaturated heptanoates can also be utilized as a nutritional supplementto overcome fatty acid metabolism deficiencies. In addition,substituted, unsaturated, and/or branched seven-carbon fatty acids whichreadily enter the mitochondrion without special transport enzymes can beutilized in the present invention. For example, 4-methylhexanoate,4-methylhexenoate, and 3-hydroxy-4-methylhexanoate are broken down bynormal β-oxidation to 2-methylbutyric acid with final degradationaccomplished via the isoleucine pathway. Likewise, 5-methylhexanoate,5-methylhexenoate, and 3-hydroxy-5-methylhexanoate are broken down bynormal β-oxidation to isovaleric acid with final degradationaccomplished via the leucine pathway.

The seven-carbon triglycerides of the present invention can beadministered orally, parenterally, or intraperitoneally. Preferably, itcan be administered via ingestion of a food substance containing aseven-carbon fatty acid source such as triheptanoin at a concentrationeffective to achieve therapeutic levels. Alternatively, it can beadministered as a capsule or entrapped in liposomes, in solution orsuspension, alone or in combination with other nutrients, additionalsweetening and/or flavoring agents. Capsules and tablets can be coatedwith sugar, shellac and other enteric agents as is known.

The method of administration is determined by the age of the patient anddegree of fatty acid metabolism deficiency. For the treatment of infantswith fatty acid metabolism defects, especially translocase deficiency,triheptanoin is preferably added as a nutritional supplement to adietary infant formula comprising low fat and/or reduced long-chainfatty acids. Exemplary commercially available infant formulas for usewith triheptanoin include Tolerex (Novartis Nutritionals, Minneapolis,Minn.), Vivonex (Ross Laboratories, Columbus, Ohio), and Portagen andPregestamil (Mead Johnson (Evansville, Ind.). Triheptanoin is added tothe formula at a concentration effective for achieving therapeuticresults. For children and adult patients requiring a nutritionalsupplement, e.g., surgery or oncology patients undergoing chemotherapy,triheptanoin is preferably supplied as a nutritional drink or as part ofa total parenteral nutrient administration.

For patients suffering from a complete breakdown of the fatty acidmetabolic pathway due to an inborn error of metabolism, triheptanoin isutilized at a concentration which provides approximately 15% to 40%,preferably 20% to 35%, and most preferably approximately 25% of thetotal calories per 24 hours.

For patients in which the fatty acid metabolic pathway is functional ata reduced efficiency (e.g., premature infant, elderly, cardiac patient),triheptanoin is utilized at a concentration which provides approximately15% to 40%, preferably 20% to 35%, and most preferably approximately 25%of the total calories per 24 hours.

Since propionyl-CoA is a metabolic by-product of triheptanoin oxidation,increased blood levels of propionic acid can result. Moreover,propionyl-CoA can enter into other enzymatic reactions which producetoxic compounds affecting the Kreb's cycle and the urea cycle.Therefore, the administration of a seven-carbon fatty acid such asn-heptanoic acid and/or triheptanoin supplement, especially in patientsexhibiting a build-up of serum propionic acid, may require theadministration of a carnitine supplement and/or a biotin and vitamin B12combination. In the presence of excess L-carnitine and the mitochondrialenzyme carnitine acetyltransferase, propionyl-CoA is converted topropionylcarnitine, a non-toxic substance which is excreted in theurine. Biotin is a vitamin cofactor required for the enzymepropionyl-CoA carboxylase which catalyzes the conversion ofpropionyl-CoA to methylmalonyl-CoA. Cyanocobalamin is a form of vitaminB12 which acts as a cofactor for the enzyme methylmalonyl-CoA mutasewhich catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA.Succinyl-CoA is readily pulled into the Kreb's cycle. Therefore, excesspropionyl-CoA in the patient's blood is removed by conversion tosuccinyl-CoA.

Example 1 Supplementation in Cell Lines

The addition of n-heptanoic acid to cultured cells (fibroblasts) takenfrom patients with a lethal form of translocase deficiency indicatedsuccessful oxidation.

Because a sibling had died at the age of four days from severetranslocase deficiency, amniocytes obtained from a fetus were examinedfor competency in fatty acid metabolism. The tests revealed that thefetus also had severe translocase deficiency.

Fibroblasts taken from the deceased sibling and amniocytes taken fromthe fetus were both evaluated for fatty acid metabolism of n-heptanoicacid (C₇) using a tandem mass spectrometry assay previously reported.(Yang, et al. 1998. “Identification of four novel mutations in patientswith carnitine palmitoyltransferase II (CPT II) deficiency,” Mol GenetMetab 64:229-236). The mass spectrometry results are presented forpalmitate in FIG. 3A and triheptanoin in FIG. 3B for the fibroblaststaken from the deceased sibling, and for palmitate in FIG. 4A andtriheptanoin in FIG. 4B for the amniocytes taken from the fetus. Resultsof the study showed that n-heptanoic acid (FIGS. 3B and 4B) wasindependent of carnitine/acylcarnitine translocase and readily oxidizedto propionyl-CoA despite the translocase deficiency in both cell lines.Based on the successful metabolism of n-heptanoic acid by the two celllines having severe translocase deficiency, the tandem mass spectrometryassay was performed on fibroblast cell lines taken from normal patientsand from patients affected by the following inherited defects of fatoxidation as proven by direct enzyme assay in other collaboratinglaboratories: carnitine palmitoyltransferase I (CPT I); severecarnitine/acyl carnitine translocase (TRANSLOCASE); carnitinepalmitoyltransferase II (CPT II); the “cardiac” form of very-long-chainacyl-CoA dehydrogenase (VLCAD-C); the “hypoglycemic” form ofvery-long-chain acyl-CoA dehydrogenase (VLCAD-H); the mitochondrialtrifunctional protein (TRIFUNCTIONAL); long-chain L-3-hydroxy-acyl-CoAdehydrogenase (LCHAD); medium-chain acyl-CoA dehydrogenase (MCAD);short-chain acyl-CoA dehydrogenase (SCAD); electron transferflavoprotein QO dehydrogenase-mild (ETF-DH mild); and electron transferflavoprotein QO dehydrogenase-severe (ETF-DH severe). Each cell line wasincubated separately with 7-²H₃-heptanoate (D3-C7), 8-²H₃-octanoate(D3-C8), 9-²H₃-nonanoate (D3-C9), and 16-²H₃-palmitate (D3-C16). Theresults are given as tandem mass spectrometry in FIG. 5A-L for D3-C7;FIG. 6A-L for D3-C8; FIG. 7A-L for D3-C9; and FIG. 8A-L for D3-C16.

The normal cell line and eleven abnormal cell lines were analyzed ingroups of three. For quantitative purposes, labeled internal standardswere included in each analysis and are designated as “IS” on the firstprofile in each group. The mass numbers for these standards are: m/z 420(²H₆-palmitate-C16), m/z 308 (²H₆-octanoate-C8), m/z 269(²H₉-isovaleric-C5), and m/z 237 (²H₅-propionate-C3), wherein m/z is themass:charge ratio.

As shown in FIG. 8A, when normal cells are incubated with D3-C16, aprofile of labeled acylcarnitine intermediates can be observed from C16down to and including C4. The mass numbers for these ²H₃-labeledacylcarnitines, as methyl esters, are m/z 417 (C16), m/z 389 (C14), m/z361 (C12), m/z 333 (C10), m/z 305 (C8), m/z 277 (C6), and m/z 249 (C4).

When you observe the various cell lines incubated with 16-²H₃-palmitateD3-C16 (FIG. 8A-L), virtually no oxidation occurs in CPT I cells (FIG.8B), and a minimal amount of palmitoylcarnitine from D3-C16 (m/z 417(C16)) is observed as expected since palmitate cannot be easilyconverted to palmitoylcarnitine for transport into the mitochondrion. Inboth TRANSLOCASE (FIG. 8C) and CPT II (FIG. 8D) deficient cell lines, nooxidation occurs but large quantities of labeled palmitoylcarnitine fromD3-C16 (m/z 417 (C16)) accumulate as a result of the presence of CPT I.The abnormal profiles of labeled acylcarnitines in VLCAD-C (FIG. 8E),VLCAD-H (FIG. 8F), TRIFUNCTIONAL (FIG. 8G), LCHAD (FIG. 8H), ETF-DH-mild(FIG. 8K), and ETF-DH-severe (FIG. 8L) cell lines reflect accumulationscorresponding to the carbon chain length specificity of the missingenzyme activity. In MCAD (FIG. 8I), oxidation clearly proceed down tothe level of C8 (m/z 305.3), at which point there is a markedaccumulation reflecting the substrate specificity of the missing MCADenzyme. Similarly, in SCAD (FIG. 8J), oxidation stops at m/z 249(²H₃-butylcarnitine-C4). These results indicate that CPT I, translocase,CPT II, VLCAD, trifunctional, LCHAD, SCAD, and ETF-DH are all requiredfor complete oxidation of palmitate.

In the case of D3-C8 (FIG. 6A-L), the relative accumulation of m/z 305(²H₃-octanoate-C8) indicates a distinct requirement for both translocase(FIG. 6C) and MCAD (FIG. 6I) for complete oxidation. While commercialmedium chain triglycerides (MCT), the major component being octanoate,are considered independent of CPT I, translocase, and CPT II, this datafor ²H₃-octanoate-C8 indicates that MCT is not an effective treatmentfor severe translocase deficiency. Further, the data illustrates thatMCT would not be appropriate treatment for MCAD deficiency.

For cell lines treated with odd-carbon substrates D3-C7 (FIG. 5A-L) andD3-C9 (FIG. 7A-L), the beneficial effect is based on: (1) the absence ofthe diagnostic profile which could be produced to some extent fromoxidation of unlabeled endogenous lipid in the culture medium; and (2)the relative amounts of m/z 235 (²H₃-propionate-C3) as the labeled endproduct of odd-carbon degradation compared to that seen in the normalcontrol cells (FIG. 5A for D3-C7 or FIG. 7A for D3-C9). This relativeamount of m/z 235 (²H₃-propionate-C3) is compared to the level of theinternal standards at m/z 269 (²H₉-isovaleric-C5) and m/z 237(²H₅-propionate-C3). For D3-C9, an increase was observed at m/z 319(9-²H₃-nonanoate) in TRANSLOCASE, CPT II, and LCHAD cell lines. Theseresults indicate that translocase, CPT II, and LCHAD are all requiredfor complete oxidation of nonanoate.

For D3-C7, the relative amounts of ²H₃-propionate-C3 (m/z 235) producedfor the normal cells and CPT I, translocase, CPT II, VLCAD,trifunctional, LCHAD, and SCAD abnormal cell lines (FIGS. 5A-H and J)are either comparable to or in excess of the amount seen in normalcells, indicating that beneficial oxidation of the precursor occurred.One observed exception is MCAD deficiency (FIG. 5I), which is expectedas D3-C7 requires MCAD for oxidation, and in its absence, m/z 291(²H₃-heptanoylcarnitine-C7) is markedly increased. For ETF-DH, nooxidation of labeled 7-²H₃-heptanoate was observed. These resultsindicate that, with the exception of MCAD and ETF dehydrogenase,n-heptanoic acid-supplemented compositions can be used in the treatmentof the following fatty acid defects: translocase deficiency; carnitinepalmitoyltransferase I and II deficiencies; L-3-hydroxyacyl-CoAdehydrogenase (LCHAD) deficiency; very-long-chain acyl-CoA dehydrogenase(VLCAD) deficiency, and short chain acyl-CoA dehydrogenase (SCAD)deficiency.

Example 2 In Vivo Utilization of Triheptanoin Supplementation in SevereTranslocase-Deficient Patient

Treatment of the infant with severe neonatal translocase deficiencyidentified in Example 1 using triheptanoin-supplemented low fat formulawas successful. Additionally, there is support for the correlationbetween the clinical response to triheptanoin therapy and the in vitromass spectrometry analysis of the infant's amniocytes.

At 38 weeks gestation, delivery of the infant whose amniocytes testedpositive for severe translocase deficiency as described in Example 1 wasaccomplished. Cord blood was analyzed for total and free carnitinelevels as well as levels of individual acylcarnitines by tandem massspectrometry. (Yang, et al. 1998. “Identification of four novelmutations in patients with carnitine palmitoyltransferase II (CPT II)deficiency,” Mol Genet Metab 64:229-236). Maternal blood at the time ofdelivery was also assayed for these same levels. Results confirmed thatthe infant suffered from severe translocase deficiency.

Within the first twelve hours after delivery, a low fat formulasupplemented with triheptanoin was fed to the infant via a nasogastrictube. Subsequent feedings with the triheptanoin-supplemented formulawere given at the same frequency as any full-term infant. Supplements ofcarnitine, biotin, and cyanocobalamin were not required.

Arterial blood gases (ABG's), electrolytes, serum urea nitrogen (BUN),creatinine, ammonia, glucose, serum creatine phosphokinase (CPK), ALT,AST, hemoglobin (Hgb), and hematocrit (Hct) were monitored according tostandard neonatal intensive care procedures. Acylcarnitines werequantified twice daily by tandem mass spectrometry. Quantitative urineorganic acid analysis was performed as well to monitor the amount ofdicarboxylic acids present in the urine.

The intervention of triheptanoin-supplemented formula was a totalsuccess in suppressing the effects of translocase deficiency. During theinfant's hospital stay, the various physiological parameters given abovewere reported within normal ranges. The infant was discharged from thehospital at 7-8 weeks of age exhibiting perfect dietary management withthe triheptanoin-supplemented formula. During continued maintenance onthe triheptanoin-supplemented formula, the infant has maintained anaverage weight gain per day of 35 grams per day, compared to the averageweight gain of 20-25 grams per day for the average formula-fed infant.At four and a half months of age, the infant continued to thrive on thetriheptanoin-supplemented formula, and no carnitine, biotin, or vitaminB12 supplements had been required.

1-16. (canceled)
 17. A composition comprising a seven carbon fatty acidsource in a dosage unit, wherein the dosage unit comprises at leastabout 15 grams of the seven carbon fatty acid source and wherein thecomposition is provided in an amount suitable for providing 15% to 40%of the daily dietary calories of a human.
 18. The composition of claim17, wherein the seven carbon fatty acid source is heptanoic acid or atriglyceride thereof.
 19. The composition of claim 17, wherein the sevencarbon fatty acid source is triheptanoin.
 20. The composition of claim17, wherein the composition is provided in an amount suitable foradministration in a range of 1.5 to 4 grams per kilogram body weight perday.
 21. The composition of claim 17, wherein the composition isprovided in an amount suitable for administration in a range of 1.5 to 3grams per kilogram body weight per day.
 22. The composition of claim 17,wherein the dosage unit comprises 15 to 75 grams of the seven carbonfatty acid source.
 23. The composition of claim 17, wherein the dosageunit comprises 75 grams of the seven carbon fatty acid source.
 24. Thecomposition of claim 17, wherein the composition is provided in anamount suitable for providing 20% to 35% of the daily dietary caloriesof a human.
 25. The composition of claim 17, wherein the composition isprovided in an amount suitable for providing 25% to 35% of the dailydietary calories of a human.
 26. The composition of claim 17, whereinthe composition is provided in an amount suitable for providing 30% ofthe daily dietary calories of a human.
 27. The composition of claim 17,wherein the composition is provided in an amount suitable for providing25% of the daily dietary calories of a human.
 28. The composition ofclaim 17, wherein the composition is a nutritional supplement.
 29. Thecomposition of claim 17, wherein the composition is a dietaryformulation.
 30. The composition of claim 17, wherein the composition isa pharmaceutical composition.
 31. The composition of claim 17, whereinthe composition is administered parenterally.
 32. The composition ofclaim 17, wherein the composition is administered enterally.
 33. Thecomposition of claim 17, wherein the composition is administered orally.